Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development
© Appel et al; licensee BioMed Central Ltd. 2001
Received: 7 April 2001
Accepted: 16 July 2001
Published: 16 July 2001
Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling.
Zebrafish embryos express multiple delta and notch genes throughout the developing nervous system. All or most proliferative precursors appeared to express notch genes whereas subsets of precursors and post-mitotic neurons expressed delta genes. When we ablated identified primary motor neurons soon after they were born, they were replaced, indicating that specified neurons laterally inhibit neighboring precursors. Mutation of a delta gene caused precursor cells of the trunk neural tube to cease dividing prematurely and develop as neurons. Additionally, mutant embryos had excess early specified neurons, with fates appropriate for their normal positions within the neural tube, and a concomitant deficit of late specified cells.
Our results are consistent with the idea that zebrafish Delta proteins, expressed by newly specified neurons, promote Notch activity in neighboring precursors. This signaling is required to maintain a proliferative precursor population and generate late-born neurons and glia. Thus, Delta-Notch signaling may diversify vertebrate neural cell fates by coordinating cell cycle control and cell specification.
Specification of cells at different times and places is critical for generation of cellular diversity in the vertebrate nervous system. Distinct types of neurons and glia develop at characteristic times and places  and molecular signals that promote formation of different cell types are regulated spatially and temporally . Thus, neural pattern formation requires coordination of signals that provide spatial and temporal information.
Lateral inhibition, or lateral specification, is one process by which fine patterns of distinct cell types are generated [3, 4]. Among cells that have the potential to adopt the same fate, lateral inhibition specifies some cells for a primary or preferred fate and others for a secondary or alternative fate. Cell ablation experiments are used to identify lateral inhibition: when cells that would normally adopt a primary fate are removed, cells that would otherwise acquire a secondary fate replace them. For example, when neuroblasts of the grasshopper central nervous system were ablated, they were replaced by cells normally fated to give rise to epidermis . Similar demonstrations performed in other insects , leeches , nematodes , ascidians , and zebrafish neural crest  indicate that lateral inhibition operates in diverse developmental contexts throughout metazoan development.
The molecular mechanism of lateral inhibition is rooted in interaction of the ligand, Delta, and its receptor, Notch . Various lines of evidence indicate that Delta, expressed by specified cells, activates Notch in neighboring cells directing them into alternative developmental pathways. The genetic demonstration that Delta-Notch signaling regulates insect neuroblast formation has been performed using Drosophila melanogaster: embryos lacking Delta or Notch function develop excess neural cells at the expense of epidermis .
In the nervous system of vertebrate embryos, Notch gene expression generally is associated with proliferative zones of undifferentiated cells [13,14,15,16,17,18,19,20,21]. Scattered cells within proliferative zones express Delta [18, 20, 22,23,24], although, in chick, cells in S-phase of the cell cycle do not express Delta1 suggesting that expression is limited to newly post-mitotic cells [18, 20, 23]. In frog and zebrafish neural plate, Delta gene expression appears to precede expression of early neuronal markers [25,26,27,28]. However, whether this indicates that proliferative cells of anamniote nervous systems express Delta genes is still an open question.
Newly-specified neurons may use Delta to signal to unspecified neighboring precursor cells via Notch. Such signaling could diversify neural cell fate by maintaining a pool of precursor cells that respond to other instructive signals or by promoting alternative developmental pathways. Gain-of-function experiments provide evidence for both ideas. For example, overexpression of a constitutively active form of Notch (Nac) in frog embryos led to an increase in the amount of neural tissue, even when cell division was blocked . It was suggested that this excess neural tissue arose from specification of excess uncommitted precursor cells to a neural fate once levels of the overexpressed Notch protein fell. Frog retinal cells forced to express Nac maintained a neuroepithelial morphology rather than undergoing differentiation, which also suggests that Notch activation maintains cells in an undifferentiated state . Similarly, overexpression of Delta proteins, which presumably activate endogenous Notch signaling, prevented retinal cell differentiation in frog and chick [20, 24] and formation of the earliest-born neurons in frog and zebrafish [25,26,27,28]. Each of these observations is consistent with the notion that Notch activity maintains cells in a proliferative, unspecified state.
More recently, several reports have raised the possibility that Notch activity promotes specification of certain cell types, particularly glial cells. Forced expression of Nac promoted formation of cells expressing markers of Müller glia in rat and zebrafish retinae [30, 31]. As Müller glia are among the last retinal cell types born, these results are consistent with the idea that Notch activity delays differentiation. However, Notch activity did not promote expression of markers of other late-born cell types in rat  and appeared to inhibit, rather than promote, cell proliferation in zebrafish . Additionally, Nac expression in the mouse forebrain promoted formation of radial glia, the earliest specificied cell type in forebrain  and either permitted  or promoted gliogenesis in the peripheral nervous system .
By contrast, disruption of Notch signaling results in formation of excess early-born neurons. Antisense Notch RNA treatment or expression of a dominant negative form of Notch in chick resulted in excess ganglion cells, one of the first retinal cell types to differentiate . Similarly, expression of dominant negative Delta proteins caused formation of excess early-born neurons in the retina of frog  and chick  and in the neural plate of frog  and zebrafish [26,27,28], concomitant with decreased cell proliferation  and reduced numbers of late-born neurons . Mouse embryos homozygous for a targeted mutation of Notch1 or RBP-Jκ, which encodes a downstream component of the Notch signaling pathway, had elevated levels of expression of neuronal markers, indicating formation of excess neurons . Excess neurons also developed in mouse embryos homozygous for targeted mutation of HES1, a presumptive downstream effector of Notch signaling . Recently, a mutant allele of the zebrafish deltaA (dlA) gene was identified and shown to cause formation of excess Rohon-Beard sensory neurons and decreased trunk neural crest progenitor cells [37, 38] and excess hair cells and decreased support cells in the ear .
To investigate further the role of Notch-mediated cell-cell signaling in vertebrate neural development we have tested neural pattern formation in zebrafish embryos. We show by ablating single, identified primary motor neurons that these cells normally inhibit nearby cells from adopting the primary motor neuron fate. Our experiments lead us to conclude that in the absence of lateral inhibition the time during which neighboring cells can replace a missing cell is transient. This suggests that the temporal and spatial cues that specify neural cell fate are tightly regulated. We also show that in dlA mutant embryos neural precursor cells prematurely cease dividing and differentiate as early-born neurons with identities appropriate for their positions within the spinal cord and that mutant embryos have reduced numbers of late-born neurons and glia. These observations support the idea that Delta-Notch mediated lateral inhibition coordinates transition of proliferative neural precursors into specified neurons and glia of the spinal cord.
Delta-Notch pathway genes are expressed in neural precursor cells
As a step toward a fuller understanding of the role that Delta-Notch signaling plays in patterning the zebrafish neural tube, we compared expression of several delta and notch genes to the distribution of neural precursor cells and differentiating neurons. We use the term, neural precursor cell, to indicate proliferative neuroepithelial cells that did not express Hu proteins ; we identified proliferative cells by their incorporation of BrdU. Conversely, we categorized those cells that expressed Hu proteins as post-mitotic, differentiating neurons. We examined the RNA expression patterns of delta and notch genes using a sensitive fluorescent detection method, choosing as our timepoint 24 h (hours of development at 28.5°C; ). At this stage, primary neurons have differentiated and some secondary neurons are postmitotic and have differentiated while others remain in the cell cycle . The assays described here do not discriminate between primary and secondary neurons.
We showed previously that post-mitotic primary neurons express dlA transiently at the 4 somite stage . Many neural plate cells in excess of known post-mitotic neurons expressed dlA and dlD, suggesting that these cells include neural precursors [26,27,28]. By contrast, dlB expression appeared to be limited mostly to post-mitotic neurons . At 24 h, cells throughout the trunk neural tube, including Hu-positive cells, expressed dlA (Figure 1J,K,L). Both Hu-positive and Hu-negative cells also expressed dlB (Figure 1M,N,O), however, apparently fewer of each class expressed dlB at high level than dlA. Some Hu-positive and many Hu-negative cells expressed dlD (Figure 1P,Q,R). Together, these observations suggest that a subset of neural precursors and cells specified for neuronal development express dlA, dlB and dlD.
Lateral inhibition regulates primary motor neuron number
Widespread neural expression of delta and notch genes is consistent with the possiblity that cell-cell signaling contributes to pattern formation within the zebrafish nervous system. To examine this directly, we performed experiments designed to test whether lateral inhibition patterns primary motor neurons. Zebrafish primary motor neurons are distributed with a striking periodicity [45, 46]. In previous studies we found that islet1 (isl1) expression, which marks primary motor neurons, was modified over time. Initially, contiguous cells in a row close to floorplate expressed isl1, whereas later, isl1-expressing cells were interspersed with cells that did not express isl1 . Currently, we cannot distinguish between the possibilities that alternating cells downregulate isl1 expression or that cells that do not express isl1 move between those that do. Nevertheless, to maintain this periodicity, isl1-negative cells must not transcribe isl1, despite being equidistant with isl1-positive cells from notochord and floorplate, presumed sources of signals that induce motor neuron development . To test whether newly born primary motor neurons inhibit neighboring cells from adopting similar fates, we ablated identified CaP and VaP motor neurons  and asked whether these cells were replaced. Normally, CaP develops in every hemisegment, midway between adjacent somite boundaries. VaP develops in about one half the hemisegments; when present it is next to CaP. Both cells express isl2 whereas other primary motor neurons do not . Previous experiments showed that when CaP was removed from 13 somite stage embryos, it was never replaced . For this experiment, we extirpated CaP and, when present, VaP from the right side of spinal hemisegment 7 (R7) in 11 somite stage embryos, 1 hour earlier in developmental time than in previous experiments . We let these embryos develop until the 19 somite stage and probed for expression of isl2. Of a total of 13 embryos in which these ablations were performed, 11 developed a CaP or CaP and VaP in the ablated position, indicating that the ablated cells were replaced. The remaining two embryos showed no replacement. Although we do not know the normal fates of the cells that replaced ablated primary motor neurons, these observations are consistent with the possibility that, during normal development, primary motor neurons inhibit neighboring cells from adopting a primary motor neuron fate.
deltaAmutant embryos have fewer neural precursor cells and excess neurons
deltaAmutant embryos have excess early-specified neurons and fewer late-specified neurons and glia
We also investigated whether other cell types were affected in mutant embryos by examining expression of markers of various interneurons, including lim1, lim5 and Pax2. Spinal cord cells begin to express lim1 and pax2 by about 14–15 h [53, 54] and lim5 by 24 h  indicating that these cells are born relatively early in neurogenesis. Excess numbers of cells expressed each of these markers in dlA dx2 mutant embryos (Figure 5G,H,I,J,K,L). Notably, the dorsoventral position of cells expressing each of these markers was similar in mutant and wild-type embryos. Finally, dlA dx2 mutant embryos had greatly reduced numbers of presumptive radial glia cells, labeled by antibody zrf-1 (Figure 5M,N). Although we do not know the time or place that these cells originate in the neural tube, we do not detect expression with this marker until 30–36 h, well after the onset of primary neuron birth at about 11 h and secondary motor neuron birth at about 16 h. Together with the above BrdU results, these observations indicate that disruption of Delta-Notch signaling causes excess neural precursors to be specified for early-born neuronal fates at the expense of later-born neuronal and glial fates, without interfering with mechanisms that specify early-born neuronal identity along the dorsoventral axis.
In zebrafish, both neural precursors and neurons express notch and deltagenes
Expression of activated Notch proteins can prevent neural cell differentiation [15, 16, 20, 24, 25, 29]. In vertebrate embryonic nervous systems, proliferative undifferentiated cells express Notch genes [13,14,15,16,17,18,19, 21, 24]; thus, Notch activity may maintain the neural precursor state. Notably, some differentiated neural cells, such as Müller glia and radial glia, express Notch genes [30, 32] raising the possibility that Notch signaling does more than simply block differentiation. By contrast, only post-mitotic neural cells have been shown to express Delta genes [18, 20, 23]. This pattern of expression is consistent with the idea that specified, post-mitotic cells use Delta signaling to prevent neighboring cells from differentiating. However, it differs from the fly embryonic central nervous system wherein all cells of the prospective neuroectoderm express Delta with the exception of delaminated neuroblasts . This indicates that, in flies, precursors express Delta and that those specified for neuroblast development downregulate its expression.
Here, we show that some zebrafish neural cells in S phase of the cell cycle and post-mitotic differentiating neurons express delta genes. We interpret these data to mean that a subset of proliferative neural precursors upregulate delta gene expression. Upregulation might engage a feedback loop mechanism, in which Notch activation in neighboring cells maintains them in a proliferative, unspecified state whereas those cells that upregulate Delta expression, and have decreased Notch activity, are driven to exit the cell cycle and to differentiate.
Zebrafish primary motor neurons may inhibit primary motor neuron fate in neighboring cells
Lateral inhibition was originally defined by functional tests in which ablation of a cell of primary fate allowed an adjacent cell to take that fate, but ablation of a cell of secondary fate had no effect . Ablation experiments performed on grasshoppers showed that neuroblasts laterally inhibit neighboring cells from also developing as neuroblasts . Genetic analyses of fly development then led to the view that lateral inhibition is mediated by interaction of Delta and Notch proteins expressed on neighboring cells . Our ablation studies, coupled with our studies of Delta and Notch expression and with analysis of a dlA mutation, provide evidence consistent with the possibility that similar mechanisms operate during specification of individual neural fates in a vertebrate nervous system.
Mutant and gene overexpression phenotypes in various vertebrate model systems support the notion that Delta-Notch signaling mediates lateral inhibition in the central nervous system [16, 20, 24,25,26,27,28,29, 35]. However, to our knowledge, until now direct evidence for lateral inhibition in the vertebrate central nervous system has not been reported. Previously, when primary motor neurons were ablated in 13 somite stage embryos, approximately 6 hours after birth, they were not replaced . Here, we ablated primary motor neurons 1 hour earlier in developmental time and found that, in nearly every case, they were replaced. Together, our ablation results reveal an important feature of vertebrate neuronal specification: that the mechanisms that specify fate must be exquisitely timed. We view the failure to replace primary motor neurons ablated at 13 somite stage as indicating that the time at which a cell can be specified for that fate has passed, either because neighboring precursors have been specified for other fates or because the signals that specify primary motor neurons are no longer present. Future experiments, aimed at learning the fates of cells neighboring primary motor neurons may distinguish between these possibilities.
Delta-Notch signaling regulates cell proliferation and diversifies neural cell fate
Here we took advantage of the fact that dlA dx2 mutant zebrafish embryos survive several days after onset of neuronal differentiation to investigate how Delta-Notch signaling influences neural cell fate. We show that mutant embryos have a significant reduction in proliferative neural cells and a concomitant increase in neurons. These neurons have fates characteristic of the earliest-born neurons in zebrafish. Strikingly, the molecular identities of the excess, early-born neurons in dlA dx2 mutant embryos are appropriate for their positions in the trunk neural tube. This is evident across the dorsoventral axis of the neural tube as well as within localized areas of the ventral neural tube occupied by distinct motor neurons. In other words, early-born neurons are specified appropriately in dlA dx2 mutant embryos; what these embryos lack is the ability to regulate the number of cells specified for any particular fate.
We also showed that dlA dx2 mutant embryos have reduced numbers of later-born cell types including secondary motor neurons and radial glia. One possible explanation for this effect is that the premature exit of neural precursor cells from the cell cycle depletes the precursor pool. In this view, Delta-Notch signaling diversifies neural cell fate by guiding the orderly, and prolonged, transition from neural precursor cells to specified cells. Specification of a particular cellular identity might depend entirely upon the influence of instructive signals that change with time. For example, zebrafish ventral neural precursors might be specified differently by different lengths of exposure to Hedgehog (Hh) signaling. Those competent to respond to brief exposure to Hh might be specified as primary motor neurons whereas those subject to prolonged exposure prior to differentiation might develop as secondary motor neurons . Notch activity might be important for specification of secondary motor neurons by holding them in an unspecified state for long enough to receive sufficient exposure to Hedgehog signaling. Disruption of Notch signaling would then allow an excess of precursor cells to respond promptly to Hedgehog signaling, producing an excess of primary motor neurons and a concomitant deficit of secondary motor neurons, precisely the phenotype of dlA dx2 mutant embryos we show here.
A second possibility, supported by recent publications, is that Notch activity specifies certain cell fates, in particular, glial fates. Notch activity promotes gliogenesis in forebrain, retina, and cultured neural crest [30,31,32, 34]. Our observation that dlA dx2 mutant embryos lack radial glia of the trunk neural tube also is consistent with this possibility. In fact, Notch activity might both hold neural precursor cells in an unspecified state and subsequently promote a subset of them to develop as glia. Conditional misexpression of activated Notch in individual precursor cells of the trunk neural tube might provide a way to distinguish between these possibilities.
In zebrafish, at least three notch genes are expressed by proliferative neural precursors and at least three delta genes are expressed by subsets of proliferative precursors and post-mitotic neurons. Thus, precursors fated to exit the cell cycle and differentiate may express elevated levels of delta genes, causing them to signal to neighboring notch-expressing precursors. Signaling between neighboring cells appears to regulate primary motor neuron specification: when we removed primary motor neurons soon after birth they were replaced, presumably by neighboring precursors. Genetic disruption of Delta-Notch signaling causes precursors to prematurely terminate cell division and differentiate as early-born neurons, with identities appropriate to their cell body positions, at the expense of later born neurons and glia. We propose that in the zebrafish central nervous system cell-cell interactions, mediated by Delta-Notch signaling, help coordinate transition of proliferative precursors to specified neurons and glia.
Materials and methods
Embryos from the University of Oregon and Vanderbilt University laboratory colonies were raised at 28.5°C. Staging was according to Kimmel et al. (1995). The dlA dx2 allele is incompletely penetrant and variably expressive [37,38,39]. Mutant embryos shown here are those exhibiting the most severe mutant phenotypes.
Individual CaP or CaP and VaP motor neurons were removed from the right side of segment 7 (R7) of 11 somite stage embryos by aspiration with a micropipette broken manually to a tip diameter of 8–12 μm, using methods described previously .
BrdU Labeling, in situ RNA Hybridization and Immunohistochemistry
Manually dechorionated embryos were labeled with BrdU by incubating them for 20 minutes on ice in a solution of 10 mM BrdU and 15% DMSO in Embryo Medium . The BrdU solution was replaced with Embryo Medium and the embryos were incubated for 20 minutes at 28.5°C. The embryos were then anesthetized using 3-aminobenzoic acid ethyl ester and fixed in 4% paraformaldehyde. in situ RNA hybridization on BrDU-labeled embryos was carried out as described previously  except that the probes were not hydrolyzed. Following color development, the embryos were treated with 2 N NaOH for 1 hr, washed with PBSTx (phosphate buffered saline with 0.01% TritonX-100), blocked 1 hr in PBDTx (PBS, 1% DMSO, 0.01% TritonX-100 plus 2% sheep serum and 2 mg/ml BSA, then incubated 2 hr with anti-BrdU (monoclonal G3G4, Developmental Studies Hybridoma Bank). Detection was carried out using a biotinylated goat anti-mouse secondary antibody followed by streptavidin conjugated to peroxidase (Jackson ImmunoResearch Laboratories, Inc.). A brown precipitate was formed using Fast Dab (Sigma).
For fluorescent detection of RNA hybridization and antibody labeling, embryos were first treated for RNA hybridization and then sectioned using a cryostat microtome. The sections were incubated with monoclonal 16A11 anti-Hu antibody (University of Oregon Monoclonal Antibody Facility) and anti-digoxygenin-AP antibody (Roche). Anti-Hu labeling was detected using goat anti-mouse Alexa 488 antibody (Molecular Probes) and anti-digoxygenin-AP labeling was detected using HNPP Fluorescent Detection (Roche). Images were collected using a Spot camera (Diagnostic Instruments, Inc.) mounted on an Olympus AX70 microscope. Double-label images were created using Photoshop to merge individual red and green images.
Additional in situ RNA hybridizations were performed as described previously . Additional primary antibodies used were zn-8 to detect Dmgrasp and zrf-1 (Developmental Studies Hybridoma Bank) and polyclonal rabbit anti Pax-2 (Berkeley Antibody Co.).
Thanks to C. Kimmel and R. Cornell for comments on the manuscript, M. Lardelli, J. Campos-Ortega, R. Toyama and I. Dawid, P. Blader and U. Strahle, and C. Haddon and J. Lewis for providing probes, J. Doll for help with fluorescent in situ RNA hybridization, R. Cornell for help with the ablation experiments, A. Swaims for sectioning, and members of the University of Oregon and Vanderbilt University zebrafish facility staffs for fish care. Antibody G3G4, developed by S.J. Kaufman, and antibodies zn-8 and zrf-1, developed by B. Trevarrow, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. This work was supported by NIH grants NS23915 and HD22486 to J.S.E. and Vanderbilt University Start-Up Funds and NIH grant HD38118 to B.A. Additional support for the University of Oregon zebrafish facility was provided by NIH G20-RR11724, NSF STI-9602828, the MJ Murdock Charitable Trust and the WM Keck Foundation.
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